Project Summary/Abstract Lipophagy, the selective autophagy of lipid droplets (LDs), is an essential mechanism that regulates the intracellular lipid metabolism in most eukaryotic cells. Lipophagy is accomplished by the delivery of LDs from the cytosol to the lytic compartment (lysosome in mammals or vacuole in yeast). As in other autophagic pathways, the core autophagic machinery forms the autophagic isolation membrane that sequesters the LD from the cytosol. However, how this autophagic membrane recognizes the LD when lipophagy is induced is unknown. Also, it is not clear how lipophagy is kept in check the rest of the time. Therefore, lipophagy selectivity and regulation are the key gaps in our understanding of this autophagic pathway. The mechanistic understanding in these areas is critical for the precise control of lipophagy in humans for the prevention and treatment of a whole plethora of lipid accumulation diseases, including fatty liver, metabolic syndrome of aging, obesity and atherosclerosis. Recently, we discovered a novel negative regulator of lipophagy 1 (Nrl1) that specifically represses lipophagy but not non-selective autophagy in yeast. Our preliminary data suggest that the function of Nrl1 is conserved from yeast to mammalian cells and that it promotes the accumulation of LDs in murine macrophages. Since a specific suppressor of lipophagy would be such a good therapeutic target for upregulation of the pathway in so many disease states, the first project of our research program will be dedicated to the regulation of lipophagy by Nrl1. We will study the molecular mechanism of lipophagy repression by Nrl1, how specific it is for the lipophagy pathway and the impact of Nrl1 on lipid metabolism at both the cellular (yeast and macrophages) and organismal (zebrafish) levels. The second project of our research program will be focused on the selectivity mechanisms of lipophagy. By tracking down the regulatory effects of Nrl1, we will identify its effectors, the lipophagy-specific selectivity factors. We will also extend the selectivity studies to the LD proteome and identify the selectivity factors that might be regulated in an Nrl1- independent fashion. Comprehensive analyses of the lipophagy-specific factors is an important challenge to be addressed, since such factors define the lipophagy pathway, can be used to control it and are expected to be the new autophagic proteins. The evolutionary conserved lipophagy factors will be studied further at both the cellular (yeast and macrophages) and organismal (zebrafish) levels. We will generate lipophagy-deficient zebrafish models that together with the nrl1 model of constitutive lipophagy will constitute new valuable tools to address the specific contribution of lipophagy to various lipid accumulation diseases.